The long-term goal of this research is to understand the unique role of proteins encoded by the major histocompatibility complex (MHC) in T cell recognition and activation. The murine cytotoxic T cell response to allogeneic class I and II MHC molecules is about 50 times greater than the response to xenogeneic human MHC molecules, and this has been shown to be due to species-specific structural differences between murine and human homologs. Results obtained in the last funding period have shown that positive selection of human class I specific murine T cells is enhanced in transgenic mice expressing a chimeric class I molecule with a murine alpha3 domain, suggesting that a reduced efficiency of interaction between the human alpha3 domain and murine CD8 contributes to the low xenogeneic response. Nonetheless, human class I specific responses in these mice remain well below those involving murine MHC molecules. In addition, T cell responses to chimeric class I molecules show directly that species-specific structural differences in the alpha1+2 domains are at least as important as those in alpha3 in contributing to the weak xenogeneic response. The basis for this difference is still poorly understood, and we propose three distinct approaches to garner additional information. Specific aim 1 will concentrate on the identification of residues in the alpha1+2 domains that contribute to the difference in allogeneic and xenogeneic responses. Specific aim 2 will assess the impact of species-specific structural differences in the alpha1+2 domains on positive and negative selection in transgenic mice expressing chimeric class I molecules. Specific aim 3 will test the hypothesis that CD8 interacts in a species specific way with structures in the alpha1+2 domains, in addition to those in alpha3. Specific aim 4 will test the hypothesis that the specificity for structural features common to murine class I alpha1+2 domains that are altered on human class I molecules is due to species specific differences in TCR V regions. Finally, specific aim 5 will take advantage of our previous observations on the species-specificity of CD8-alpha3 domain interactions to map the class I binding site on the CD8 molecule. The successful understanding of the reasons for the weak xenogeneic response has several important implications beyond the apparently rather esoteric question of why mice fail to mount significant T cell responses to human MHC molecules. The results will provide fundamental structural information on the interactions of class I molecules with both CD8 and TCRs, the importance of these interactions during the selection of the T cell repertoire, and whether there is a preexisting focus of TCR on MHC molecules. These studies will also contribute to an understanding of the recognition of xenoantigens in transplant situations, a topic of increasing clinical relevance. Finally, successful completion of these studies is a necessary preliminary to future studies utilizing both human CD8 and human class I transgenic mice to create mice with "humanized" immune systems that are preclinical models for vaccine testing and disease study. The proposed experiments will provide a foundation for understanding both the potential and limitations of this model system.